The long-term objective of this project is to utilize the yeast S. cerevisiae as a model system to determine the mechanism by which the cytosolic Hsp40s Sis1 and Ydjl act with Hsp70 Ssal (Ssal) to protect cells from denaturing physiological stresses such as heat-shock. The hallmark of the heat-shock response is the rapid induction of chaperone protein expression and the global suppression of protein synthesis. These events occur to prevent the accumulation of non-native proteins and suppress protein aggregation. Hsp40s function to suppress protein aggregation by binding non-native proteins and delivering them to the polypeptide binding site of HspT0. In aim 1, we propose a series of experiments that are designed to determine the mechanism for substrate binding by Hsp40s. In aim 2, we will investigate how denatured proteins that are bound to Hsp40 are delivered to Hsp70 to facilitate protein refolding. An additional feature of Hsp40 family members is that they have evolved to have different domain structures and can target Hsp70 to catalyze different cellular reactions. We have demonstrated that the Ydjl and Sis1 function in the cytosol to direct Ssal to facilitate different aspects of protein metabolism. In Aim 3 and Aim 4 we propose a series of domain swap experiments between Ydjl and Sis1 that are designed to define the rules by which Hsp70s cellular functions are specified.The outcome of these studies will define basic mechanisms by which Hsp40s function with Hsp70 to protect cells from physiological stress.